Results of plasma microparticles (MPs) measurements reported in the literature vary widely. This is clearly not only related to the lack of well-standardised MP assays, but also to variations in pre-analytical conditions. In this review we will discuss the pre-analytical variables related to plasma and MP preparation which may affect MP analysis. Additionally we will address several analytical issues in commonly used MP assays and briefly discuss some novel approaches for the detection and characterisation of MPs. Ideally MP measurements should be performed in plasma, freshly prepared directly after blood withdrawal. As platelet contamination seems to be one of the major pre-analytical problems in processing plasma for MP measurement, the use of platelet-free plasma may be preferred. When frozen-thawed plasma is used, especially PMP and annexinV-positive MP counts should be interpreted with caution. When flow cytometry is chosen as a method for quantification of MPs, some analytical conditions should be standardised, e.g. settings of the flow cytometer, quality of the antibodies, and use of counting beads. Fluorescence-nanoparticle tracking analysis and atomic force microscopy can accurately count nanosized MPs, but unfortunately the operational procedures of both methods are still time consuming and they give no information on the functional properties of MPs. The MP-TF activity assay provides information on MPs carrying active TF, regardless of their parental origin. Ultimately, standardisation of pre-analytical procedures and the introduction of reliable and rapid methods for the measurement of MPs are urgently needed to facilitate their use as biomarker in the pathophysiology of diseases.

IntroductionExtracellular vesicles (EV) are phospholipid bilayer-enclosed vesicles recognized as new mediators in intercellular communication and potential biomarkers of disease. They are found in many body fluids and mainly studied in fractions isolated from blood plasma in view of their potential in medicine. Due to the limitations of available analytical methods, morphological information on EV in fresh plasma is still rather limited.ObjectivesTo image EV and determine the morphology, structure and size distribution in fresh plasma by cryo-electron microscopy (cryo-EM).MethodsFresh citrate- and ethylenediaminetetraacetic acid (EDTA)-anticoagulated plasma or EV isolated from these plasmas were rapidly cryo-immobilized by vitrification and visualized by cryo-EM.ResultsEV isolated from fresh plasma were highly heterogeneous in morphology and size and mostly contain a discernible lipid bilayer (lipid vesicles). In fresh plasma there were 2 types of particles with a median diameter of 30 nm (25–260 nm). The majority of these particles are electron dense particles which most likely represent lipoproteins. The minority are lipid vesicles, either electron dense or electron lucent, which most likely represent EV. Lipid vesicles were occasionally observed in close proximity of platelets in citrate and EDTA-anticoagulated platelet-rich plasma. Cryo-electron tomography (cryo-ET) was employed to determine the 3D structure of platelet secretory granules.ConclusionsCryo-EM is a powerful technique that enables the characterization of EV in fresh plasma revealing structural details and considerable morphological heterogeneity. Only a small proportion of the submicron structures in fresh plasma are lipid vesicles representing EV.

Multiple myeloma (MM) is associated with an increased risk of venous thromboembolic (VTE) complications. Aim of this study was to measure microparticle-associated tissue factor (MP-TF) activity in patients with newly diagnosed MM before and after chemotherapy and to investigate whether MP-TF activity is associated with VTE. MP-TF activity was assessed in 122 newly diagnosed MM patients who were eligible for combination chemotherapy. MP-TF activity levels (17.6 fM Xa/min [8.6-33.2] (median [IQR]) were higher in untreated MM patients compared to normal healthy volunteers (4.1 fM Xa/min [2.3-6.6], p <0.001). MP-TF activity prior to the start of treatment was not different between patients who developed a VTE during follow-up (n=15) and those who did not (n=107). In 75 patients in whom plasma was obtained before and after chemotherapy, MP-TF activity decreased significantly (from 17.4 [10.2-32.8] to 12.0 [7.0-18.5] fM Xa/min, P=0.006). MP-TF activity remained, however, elevated in patients who developed VTE (15.1 [10.3-25.2]), in contrast to patients not developing VTE (11.4 [7.0-25.2], P<0.001). In conclusion, MP-TF activity is increased in patients with MM. Whether MP-TF activity has a pathogenetic role in VTE in MM patients remains to be established in future studies.

Increased microparticle tissue factor (TF) activity is not only found in cancer patients, but also in patients with cardiovascular and inflammatory diseases. Methods such as flow cytometry and impedance-based flow cytometry allow the analysis of microparticle subsets but provide no insight on which microparticles carry active TF. Conversely, the microparticle-TF activity itself does not reveal the cellular origin of the microparticles carrying the active TF.For this reason, we developed an immuno-magnetic bead method to capture subsets of microparticles directly from plasma. The method was optimized for capture of platelet-derived microparticles (PMPs) from plasma. Only 100 μl platelet-poor plasma (PPP) was needed in combination with 135 μl (27 μg) of biotinylated antihuman CD41 monoclonal antibody (MoAb) and 200 μl of streptavidin beads to achieve complete separation of PMPs from plasma. As a control, biotinylated mouse IgG1 isotype control MoAb was used instead of the anti-CD41 MoAb. Using biotinylated anti-CD14 MoAb, CD14-positive microparticles were captured from normal plasma spiked with microparticles isolated from the supernatant of lipopolysaccharide-stimulated monocytes (MoMPs). TF activity was found both in the positive (selected) and negative (depleted) fractions indicating that both CD14-positive and negative MoMPs carry active TF. We propose that this method can be used in the future to investigate the source of microparticles carrying active TF in plasma of patients with cancer and other diseases.

Background: Multiple myeloma (MM) is associated with an increased risk of venous thrombo-embolic complications. The pathogenesis of venous thromboembolism (VTE) in MM is not yet clarified. Recently an association between microparticle-associated tissue factor (MP-TF) activity in plasma and thrombosis in patients with adenocarcinoma has been reported. Aim of this study was to measure MP-TF activity in patients with MM before and after treatment with chemotherapy and to investigate whether MP-TF activity is associated with VTE in these patients. Patients and results: MP-TF activity was assessed in 123 patients with newly diagnosed multiple myeloma who were eligible for combination chemotherapy and in 20 healthy volunteers. MP-TF activity levels were higher in untreated MM patients compared to the healthy volunteers (17.8 fM Xa/min [8.8–32.8] (median [Inter Quartile Range] versus 4.7 fM Xa/min [2.3–6.6], p&lt;0.001). After three courses of chemotherapy the level of MP-TF activity had decreased significantly to 11.8 fM Xa/min [7.0–18.3], but were still higher than in controls (P&lt;0.001). VTE occurred in 12 patients (10%). In MM patients with VTE the level of MP-TF activity remained elevated after induction chemotherapy (17.9 [9.3– 36.3]), in contrast to MM patients not developing VTE (11.1 [6.9–16.6] ). MP-TF levels after chemotherapy were significantly associated with the risk of VTE, with a 4% increase of VTE risk for every fM Xa/ min MP-TF activity increase (95% CI: 1–8%, p=0.03) Conclusion: MP-TF activity levels in plasma are increased in patients with multiple myeloma and they decrease significantly after induction chemotherapy. Persistent high MP-TF activity is observed in patients who developed VTE during the induction phase chemotherapy, and is associated with VTE risk. Our findings suggest that MP-TF activity levels may have a pathogenetic role in VTE in multiple myeloma patients.